WO2002072845A2 - Salmonella promoter for heterologous gene expression - Google Patents
Salmonella promoter for heterologous gene expression Download PDFInfo
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- WO2002072845A2 WO2002072845A2 PCT/GB2002/001098 GB0201098W WO02072845A2 WO 2002072845 A2 WO2002072845 A2 WO 2002072845A2 GB 0201098 W GB0201098 W GB 0201098W WO 02072845 A2 WO02072845 A2 WO 02072845A2
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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Definitions
- This invention relates to promoter sequences which promote the expression of a suitable polynucleotide.
- Attenuated microorganisms As vaccines. It is also proposed that attenuated microorganisms may be useful for the delivery of therapeutic agents.
- the ssaG gene is a component of the Salmonella pathogenicity island SPI-2 (WO96/17951).
- Valdivia etal., Science, 1997; 277 (5334): 2007-2011 describes a promoter trap experiment to identify bacterial genes that are preferentially expressed in a host cell.
- the ssaH gene (now known as ssaG) is identified as a gene that is expressed when Salmonella typhimurium infects a host's macrophage cells. It is disclosed that the ssaH (ssaG) promoter is fused with the gene for green fluorescent protein (GFP) and placed on a multicopy plasmid system to establish whether expression occurs, thereby determining whether the ssaH gene would be expressed on infection.
- GFP green fluorescent protein
- a construct comprises the ssaG promoter, or a functional fragment thereof, operably linked to a polynucleotide heterologous to the ssaG gene.
- a Salmonella microorganism comprises a heterologous polynucleotide operably linked to the ssaG promoter.
- the ssaG promoter is used to promote expression of a polynucleotide heterologous to that of the ssaG gene and which encodes a therapeutic protein or peptide.
- a method for the expression of a heterologous polynucleotide within a Salmonella microorganism comprises integrating the polynucleotide into the microorganism's chromosome so that it is operably linked to the endogenous ssaG promoter.
- Figure 1 is a schematic representation of ssaG promoter regions cloned into S. typhimu um strains and GFP reporter vectors, wherein the arrows indicate the regions of S. typhimurium TML DNA derived from upstream of the ssaG ATG start codon;
- Figure 2 shows the level of LT-B expression in S. typhimurium strains harbouring different regions of the ssaG promoter inside macrophages;
- FIG 3 shows the serum IgG anti-LT-B responses in BALB/c mice on day 28 post-immunisation with various strains (Studyl);
- Figures 4a and 4b show respectively the serum IgG anti-LT-B responses in BALB/c mice on days 28 and 42 post-immunisation (Study 2); Figures 5a and 5b show respectively the serum IgG anti-LT-B responses in
- Figure 6 shows a FACS analysis of GFP expression in S. typhimurium strains infecting J774A-1 cells
- Figure 7 shows GFP expression in S. typhimurium strains infecting J774A-1 cells, taken at points up to 24 hours;
- Figure 8 shows LacZ expression in S. typhimurium strains harbouring GFP/LacZ reporter vector infecting J774A-1 cells, taken at points up to 24 hours.
- the present invention is based on the discovery that the ssaG promoter is surprisingly effective at promoting the expression of heterologous polynucleotldes.
- the promoter may therefore be used in therapy to drive expression of polynucleotldes encoding therapeutic proteins or peptides etc.
- references to therapy also include preventative treatments, e.g. vaccination.
- veterinary applications are also considered to be within the scope of the invention.
- the ssaG promoter is located upstream of the start codon for the ssaG gene, identified in Hensel et al. Mol. Microbiol., 1998; 30(1): 163-174.
- the sequence identified herein as SEQ ID NO. 1 contains the functional promoter and may be used as part of the invention.
- sequences identified herein may be further fragmented to obtain more defined polynucleotides comprising the promoter sequence. Synthetic or recombinant techniques may be used to generate the shorter fragments which retain the promoter function.
- the fragment may comprise at least 30 nucleotides, preferably at least 40 nucleotides and most preferably at least
- Sequence identity may be at least 50%, preferably 60% and most preferably at least 90%.
- the promoter comprises at least the sequence from nucleotide number 330 to 503 (173 bp) of SEQ ID NO. 1, more preferably at least the sequence from nucleotide number 229 to 503 (275 bp) and most preferably from nucleotide number 39 to 503 (465 bp) of SEQ ID NO. 1.
- identity is known in the art. The use of the term refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared.
- Levels of identity between gene sequences can be calculated using known methods.
- publicly available computer-based methods for determining identity include the BLASTP, BLASTN and FASTA (Atschul etal, J. Molec. Biol., 1990; 215:403-410), the BLASTX programme available from NCBI, and the Gap programme from Genetics Computer Group, Madison Wl.
- the levels of identity may be obtained using the Gap programme, with a Gap penalty of 50 and a Gap length penalty of 3 for the polynucleotide sequence comparisons.
- the promoter may be isolated and used as part of a recombinant construct or vector, for delivery into a host cell etc. Alternatively, in the context of Salmonella microorganisms, the endogenous promoter may be used to drive expression of a heterologous polynucleotide (or gene) which is inserted downstream of the promoter.
- a construct according to the invention may be in the form of an expression vector or plasmid, comprising the promoter, operably linked to the heterologous polynucleotide. Additional selection marker genes or regulatory elements may also be included as part of the construct.
- the construct may also be designed so that it is capable of integrating within a host's chromosome, for example by the utilisation of transposable elements.
- the promoter is to be used endogenously, with a heterologous polynucleotide inserted functionally downstream, then various techniques may be used to achieve this, including homologous recombination. All this will be apparent to the skilled person.
- the polynucleotide for use in the invention may be any that encodes a product that has a therapeutic utility.
- Therapeutic products are those that are useful in the treatment or prevention of a condition or disease of the human or animal body.
- the polynucleotide may encode a protein that acts at a particular receptor site. Hormones and growth factors are therefore within the scope of the invention.
- the encoded product may be antigenic, eliciting an immune response.
- the antigenic fragments (product) will be at least 10 amino acids in size, preferably at least 20 amino acids and most preferably at least 30 amino acids in size.
- Suitable antigens include: Allergy Vaccines
- B cell mlgE peptide (Tanox), human IgE decapeptide (PT), allergen peptides e.g. cat dander (fel d), house dust mite (Der p 1, Der f 1), and companion animal vaccines e.g. canine IgE peptides Cancer Vaccines
- MUC1 human mucin expressed by breast (and other) epithelial cancer cells
- EGFRvlll Variant of epidermal growth factor receptor expressed on numerous cancer cell types
- hCG peptides (Keutmann loop and CTP-37) expressed by bladder cancer cells
- idiotypic peptides expressed by human lymphoma cells
- Peptides from sperm antigens e.g. FA-1 and FA-2, lactate dehydrogenase, SP-10, NZ-1 , NZ-2
- sperm antigens e.g. FA-1 and FA-2, lactate dehydrogenase, SP-10, NZ-1 , NZ-2
- Peptides from oocyte antigens e.g. Zonula pellucida-3 Viral Vaccines
- Respiratory syncitial virus _(RSV) F and G proteins
- MV Measles virus
- F protein Measles virus
- HIV proteins/peptides (gp41 Kennedy epitope), gp 120 major immunodominant loop (concensus sequence) chemokine receptor peptides e.g. CXCR4, CCR5; nef, rev, pol, tat,
- VP1 Hepatitis A virus
- Hepatitis B virus HbcAg, HbcAg/Pre-S1 , HbsAg, S-loop peptide
- CSP Parasite Vaccines Malarial: (CSP (NANP)n, SSP-2, MSP-1, AMA-1, RAP-1, EBA 175, LSA-1),
- FnBP S. aureus proteins
- EPEC/EHEC e.g. intimim, ETEC (CFA, LT-B),
- Pertussis pertactin, FHA
- Non-typeable H. influenzae (P6 protein),
- the heterologous polynucleotide may be a therapeutic nucleic acid, e.g. one that is transcribed to produce an anti-sense RNA.
- the constructs of the invention may be used in any suitable microorganism for the delivery of the therapeutic protein, peptide, RNA etc.
- the microorganism is a Salmonella microorganism, e.g. S. typhi or S. typhimurium.
- E. coli and Shigella can also be used as the delivery vehicle by the incorporation of the ssaG promoter and heterologous gene.
- the microorganism will usually be attenuated, i.e. of reduced virulence.
- the attenuation of virulence is known to those skilled in the art and methods for preparing such microorganisms are well known.
- Virulence genes are known, and, in the context of Salmonella, include those located within SPI-2. The genes may be targetted for inactivation with the insertion of ssaG promoter: heterologous gene fusion.
- a particularly preferred attenuated microorganism is a Salmonella strain that is mutated to prevent expression of the ssaV and aroC genes. Salmonella strains disrupted in this way are disclosed in WO00/68261. The promote ⁇ gene construct may be inserted within one of the disrupted genes, e.g. aroC.
- the heterologous polynucleotide is inserted functionally downstream of the ssaG promoter in an attenuated Salmonella microorganism.
- the insertion of the heterologous polynucleotide may disrupt the ssaG gene, which may in turn result in attenuation.
- the attenuated microorganism may then be used in a vaccine preparation, with the heterologous polynucleotide further promoting the prophylactic effect.
- Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, to provide effective immunisation against infection.
- the ssaG promoter is also shown in the experiments detailed below to be highly regulated with expression limited to conditions occurring in the natural macrophage environment. This may offer advantages for the controlled expression of genes encoding toxic (e.g. cytotoxic) products.
- the promoter can therefore be used to express proteins in specific environments in vivo or in vitro. In addition, specific induction of the promoter activity may be useful for the production of proteins etc. in a fermentation process. All this will be apparent to the skilled person.
- the following Example illustrates the invention. Example
- a series of Salmonella typhimurium mutant strains were constructed to test the effectiveness of the ssaG promoter in gene expression. The strains were then tested in cell culture, in vitro and in vivo experimental models to study gene expression.
- the ssaG promoter was compared to an alternate in vivo inducible pagC promoter.
- This promoter drives expression of the pagC gene, which encodes a 188 amino acid outer membrane protein required for full virulence of S. typhimurium in mice (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93).
- pagC is a member of the PhoP/PhoQ regulon and is upregulated 77-fold in S. typhimurium infected mouse macrophages harbouring a multicopy pagC-lacZ reporter vector (Alpuche-Aranda etal., PNAS, 1992; 89(21): 10079-83).
- the pagC promoter has been investigated in several studies as an in vivo inducible promoter for the delivery of foreign antigens by S. typhimurium. Dunstan etal., Infect. Immun., 1999; 67(10): 5133-41, reported that the pagC promoter functioned more effectively than the nirB and katG in vivo inducible promoters for the delivery of antigens using a multicopy /acZ/luciferase reporter vector in S. typhimurium ⁇ aroAD strains.
- the pagC promoter integrated as a single chromosomal copy, has also been shown to enhance the immunogenicity of model antigens in mice in comparison to constitutive promoters (Hohmann etal., PNAS, 1995; 92(7): 2904-8).
- Table 1 summarises the list of strains used in this study. The associated ssaG promoter regions present in each strain and vector generated for use in this study are shown in Figure 1.
- the sseG gene lies upstream of the ssaG gene in S. typhimurium and the C-terminal portion of this protein is encoded within the ssaG promoter region shown in Figure 1.
- the intergenic region extends from base 414 to 506 of SEQ ID NO. 1 , and is illustrated in Figure 1. Therefore the 173, 275 and 465 bp ssaG promoter regions cloned into S. typhimurium RST001 , RST005 and RST012 all contain part of the sseG coding region (however none of these constructs contain the entire sseG gene).
- S. typhimurium harbouring defined mutations in ssaV and aroC are disclosed in WO00/68261.
- all S. typhimurium aroC strains were routinely cultured at 37°C in LB-aro broth (Luria Bertani broth supplemented with 10.0 ⁇ g/ml aminobenzoic acid, 40.0 ⁇ g/ml L- phenylalanine; 40.0 ⁇ g/ml L-tryptophan; 40.0 ⁇ g/ml tyrosine and 10 ⁇ g/ml 2,3- dihydroxybenzoic acid). Broth cultures were shaken at 180 rpm unless stated otherwise.
- S. typhimurium strain SL1344 was cultured in standard LB broth and the presence of the GFP reporter vectors transformed into this strain selected by the addition of 100 ⁇ g/ml ampicillin to the growth medium.
- S. typhimurium RST001, RST005, RST015 and RST012 were all derived from the attenuated S. typhimurium strain WT05, which contains defined deletions in the aroC and ssaV genes of wild-type S. typhimurium TML. Construction of WT05 is described in WOOO/68261.
- RST001 , RST005 and RST012 contain promotergene fusions that have been integrated in the S. typhimurium WT05 chromosome at the site of the attenuating aroC deletion.
- the promotergene fusions consist of a variable length of the ssaG promoter region fused to the eltB gene, which expresses the Escherichia coli heat labile toxin B subunit (LT-B).
- the ssaG promoter region corresponds to the region of DNA located immediately upstream of the ssaG gene in S. typhimurium and in this study the ssaG promoter region has been derived from S. typhimurium strain TML.
- LT-B is under the control of ssaG promoter regions of 173 bp, 275 bp and 465 bp respectively.
- three different primer pairs were used to PCR amplify these regions from the chromosomal DNA of S. typhimurium TML.
- Oligonucleotide primer pairs 450SGF/SGR, 300SGF/SGR, and 150SGF/SGR were used generate the 465, 275 and 173 bp fragments, respectively.
- the primers resulted in the incorporation of 5'Xhol and 3' Ndel sites into each DNA amplicon.
- the eltB gene was PCR amplified from E. co//07 ⁇ :H11 (American Type Culture Collection strain number 35401) using primers LTB/forward and LTB/reverse.
- the eltB amplicon which contains a 5' Ndel site and a 3' Avrll site, was subsequently ligated downstream of the 3' Ndel sites present in the three ssaG promoter amplicons and the ssaG-eltB fusions cloned into the pBluescript II KS (+) cloning vector (Stratagene) modified to contain both Xhol and Avrll sites in its multiple cloning region.
- Plasmid pMMIAC ⁇ is a pUC18 based vector, which contains a 4.8kb Hindlll fragment derived from S. typhimurium TML.
- the 4.8 kb insert harbours the S. typhimurium aroC gene into which has been engineered a defined 0.6 kb deletion.
- Xhol and Avrll restriction sites have been introduced at the 5' and 3' sites respectively of the deletion, and there is approximately 3 kb of upstream DNA and 1.7 kb of downstream DNA flanking the aroC gene.
- the nucleotide sequence of the constructs was confirmed by double strand nucleotide sequencing to ensure that the PCR steps had not introduced any errors.
- pCVD442 was selected for use as the suicide vector to deliver the modified DNA as it has previously been used to introduce defined deletions into the chromosome of Gram- negative bacteria.
- the resulting suicide constructs were then electroporated into S. typhimurium WT05.
- the pagC-eltB fusion was inserted into pMMIAC ⁇ and then transferred into the suicide vector pCVD442, in preparation for insertion into the S. typhimurium WT05 chromosome at the site of the aroC deletion.
- the ELISA assay exploits the binding of LT-B to its cognate receptor protein, monosialo-ganglioside (GM-1).
- GM-1 coating buffer 0.5 ⁇ g/ml of GM-1 (Sigma E- ⁇ 015) dissolved in 50 mM glycine, 100 mM NaCI, 0.2 mM EDTA, 50 mM NaF, 0.1 % (w/v) deoxycholate).
- the plates were then washed three times with PBST. Each well was then blocked (200 ⁇ l/well) with 3% (w/v) BSA in PBS for 1 hour at 37 °C with constant shaking.
- the plate was washed as above. 50 - 100 ⁇ l of bacterial lysate samples for GM-1 capture ELISA analysis were then added to wells and incubated for 1 hour at 37°C with constant shaking. The plates were washed with PBST and 50 ⁇ l of 0.6 ⁇ g/ml Goat anti-LT (Biogenesis 4330-1104) dissolved in PBST plus 1 % (w/v) BSA added to each well. Plates were incubated at 37 °C for 1 hour with constant shaking and then washed in PBST.
- Bound Goat anti-LT was detected by the addition of 50 ⁇ l/well of a mixture of biotinylated Rabbit anti-Goat Ig (Dako, E0466) and streptavidin peroxidase (Dako P0397), both diluted 1 in 2,500 in PBST plus 1% (w/v) BSA. Following incubation at 37°C for 1 hourwith constant shaking, the plates were washed in PBST and 100 ⁇ l/well of SigmaFast OPD detection reagent (Sigma, P-9167) added, dissolved according to the manufacturer ' s instructions. Plates were incubated for 10 minutes at room temperature and the OD 450nm recorded. Concentrations of LT-B in test samples were calculated from a standard curve prepared using purified LT (ICN Biomedicals, 151074) dissolved in PBST. LT-B concentrations were expressed as ng/ml per 10 8 cfu.
- U937 cells were selected as they are a macrophage-like cell-line, and macrophages are the in vivo site of replication for Salmonella strains.
- U937 cells were grown in 150 cm 2 tissue culture flasks containing 100 ml cRPMI (RPMI medium supplemented with 2 mM glycine, 10% (v/v) foetal calf serum (FCS), 100 units/ml penicillin, 100 ⁇ g/ml streptomycin). After 3-4 days growth at 37°C in the presence of 5% CO 2 the cells were harvested and resuspended in cRPMI to give 3.0 x 10 5 viable cells/ml.
- cRPMI RPMI medium supplemented with 2 mM glycine, 10% (v/v) foetal calf serum (FCS), 100 units/ml penicillin, 100 ⁇ g/ml streptomycin.
- the cells were differentiated by adding 100 ng/ml phorbol myristate acetate (PMA). 24 ml aliquots of cells were then dispensed into 75 cm 2 tissue culture flasks, using 4 flasks per S. typhimurium strain to be tested. The flasks were incubated at 37 °C in 5% CO 2 for 96 hours or until a confluent monolayer was formed. 24 hours prior to the addition of S. typhimurium cells, the cRPMI medium was removed from the flasks, and 24 ml of PBS added to wash each flask.
- PMA phorbol myristate acetate
- the PBS was removed by aspiration and replaced with 24 ml of RPMIg (RPMI supplemented with 2 mM glycine, 10% (v/v) FCS).
- RPMIg RPMI supplemented with 2 mM glycine, 10% (v/v) FCS.
- S. typhimurium strains were grown overnight in 20 ml of LB-aro broth, harvested and re- suspended in 20 ml of fresh LB-aro.
- the bacteria were opsonised by adding 75 ⁇ l of bacterial culture to 75 ⁇ l of human serum (human serum (minus IgA), Sigma, S5016). The samples were vortexed and incubated at room temperature for 20 minutes. 400 ⁇ l of RPMIg was then added to the cells.
- the tissue culture medium was removed from the U937 cells and replaced with 24 ml RPMIg (RPMIg supplemented with 10.0 ⁇ g/ml aminobenzoic acid, 40.0 ⁇ g/ml L-phenylalanine, 40.0 ⁇ g/ml L-tryptophan, 40.0 ⁇ g/ml tyrosine and 10 ⁇ g/ml 2,3-dihydroxybenzoic acid).
- RPMIg RPMIg supplemented with 10.0 ⁇ g/ml aminobenzoic acid, 40.0 ⁇ g/ml L-phenylalanine, 40.0 ⁇ g/ml L-tryptophan, 40.0 ⁇ g/ml tyrosine and 10 ⁇ g/ml 2,3-dihydroxybenzoic acid.
- 480 ⁇ l of opsonised cultures was added to each of the 4 flasks and the flasks incubated for 1 hour at 37 °C in 5% CO 2 . The viable counts of the bacterial
- the differentiated U937 cell count was determined by trypan blue exclusion staining.
- the culture medium was removed by aspiration and 24 ml of fresh RPMIg supplemented with 200 ⁇ g/ml gentamycin added to each flask and incubated at 37°C in 5% CO 2 for 1 hour to kill extracellular bacteria.
- the tissue culture media was again removed and replaced with 24 ml RPMIg supplemented with 16 ⁇ g/ml gentamycin.
- the culture medium was removed by aspiration and the cells washed with 24 ml of PBS.
- the U937 cells were lysed by adding 24 ml of PBS containing 1% (v/v) Triton X-100 to each flask and the samples incubated at room temperature for 20 minutes. The lysates were mixed thoroughly and harvested by centrifugation. The resulting pellets containing the S. typhimurium bacteria were resuspended in 100 ⁇ l PBS plus 0.05% (w/v) Tween-20 and incubated at room temperature for 10 minutes, with vortexing every 2 minutes. The lysates were stored at -20°C until further use. 50 ⁇ l aliquots of lysates were used in the GM-1 capture ELISA described above to quantify LT-B expression.
- LT-B by S. typhimurium from the in vivo inducible ssaG and pagC promoters was also compared in cells incubated in standard LB broth and in an intracellular salts medium (ISM) designed to approximate some of the environmental conditions experienced by Salmonella inside the host macrophage.
- ISM intracellular salts medium
- LT-B expression was measured over 20 hours and is expressed as ng LT-B produced per 10 8 cfus.
- the overall expression levels of LT-B in ISM were lower than those observed in the macrophage study described above, implying that maximal induction from the promoters is not fully achieved in ISM and thus this medium does not fully replicate the conditions inside the macrophage.
- mice Three in vivo studies were carried out to evaluate the levels of anti-LT-B IgG generated to S. typhimurium RST001, RST005, RST012 and RST015 in 6-8 week old BALB/c mice.
- ⁇ mice were dosed per group and each mouse was immunized orally on day 0 with approximately 2-4 x10 10 cfus of the relevant strain, taken from an overnight culture in LB-aro.
- study 1 mice were anaesthetized prior to immunization, whereas in studies 2 and 3 non-anaesthetized mice were used. Blood was sampled by tail bleeding at days 2 ⁇ and by exsanguination at day 42.
- Sera were prepared by adding an equal volume of SeraSieve (Hughes and Hughes Ltd.) to the blood. Clotting of the blood was then allowed to proceed at room temperature for 2 hours. The samples were centrifuged at 13000 rpm in a microcentrifuge for 10 minutes, and the supernatants (sera) collected and stored at -20° prior to ELISA analysis for anti- LT-B IgG (see method in section 2.5).
- GM-1 (Sigma, E-6015) dissolved to 30 ⁇ g/ml in 50 mM sodium carbonate buffer, pH 9.6, was used to coat wells of Dynex Immulon 4, 96-well flat- bottomed plates and incubated overnight at 4°C. The plates were then washed three times with PBST (phosphate buffered saline (PBS) containing 0.05% (w/v) Tween-20) and blocked by the addition of 200 ⁇ l/well of 3% (w/v) bovine serum albumin (BSA) in PBS. Following incubation for 1 hour at 37°C with constant shaking, the plates were washed in PBST as described above.
- PBST phosphate buffered saline
- BSA bovine serum albumin
- Results of this assay are expressed as end-point titres, which correspond to the last dilution of sera at which the OD 492nm is equal to the mean OD 492nm plus 3 times the standard deviation of the blank wells (blank well contain PBST in place of sera).
- GFP green fluorescent protein
- Table 3 shows the vectors used in this study. Table 3.
- GFP green fluorescent protein
- pJKDIO is a 6.8 kb vector which contains the GFP reporter gene derived from pGFPmut3.1 (Clontech) cloned upstream of the lacZ reporter gene from pQF50.
- Two strong terminator sequences are also cloned upstream of the GFP gene to prevent read-through from the vector.
- the ssaG promoters were cloned immediately upstream of the GFP 'ATG' start codon and the correct orientation of the inserts confirmed by PCR.
- the resulting reporter vectors p1A/1 (467 bp ssaG promoter), p1 B/1 (275 bp ssaG promoter), p1C/1 (166 bp ssaG promoter) and p1D/1 (97 bp ssaG promoter) were then transformed into S. typhimurium SL1344 by electroporation. Fluorescence activated flow cytometry (FACS) analysis of GFP expression from S. typhimurium strains infecting the mouse macrophage cell line J774A.1
- J774A.1 cells (ECACC#91051511) were cultured in DMEMg medium (Dulbeccos modified Eagles medium plus 1000 mg/L glucose (Sigma D5546) supplemented with 10% (v/v) heat-inactivated FCS (Sigma F9423), 2 mM L-glutamine (Sigma 7513) and Penicillin/Streptomycin (Sigma P0781) at a final concentration 100U/100 ⁇ g/ml). Prior to infection, J774A.1 cells were harvested and the cell count assessed by mixing cells 1 :1 with trypan blue vital stain.
- DMEMg medium Dulbeccos modified Eagles medium plus 1000 mg/L glucose (Sigma D5546) supplemented with 10% (v/v) heat-inactivated FCS (Sigma F9423), 2 mM L-glutamine (Sigma 7513) and Penicillin/Streptomycin (Sigma P0781) at a final concentration 100U/100 ⁇ g/ml).
- the cells were diluted to 2x10 5 viable cells/ml in DMEMg, and 0.5 ml volumes dispensed into each well of 24-well tissue culture plates. The plates were placed into a humidified CO 2 incubator at 37°C for two days. On the day of the infection, the medium from each well containing J774A.1 cells was aspirated and the cells washed with three volumes of DMEMg. 0.5 ml of DMEMg was then added to each well. 100 ⁇ l aliquots of bacteria (10 6 organisms per 100 ⁇ l) were added to the wells and to produce a multiplicity of infection (MOI) of 10 bacteria per J774A.1 cell.
- MOI multiplicity of infection
- GFP expression was examined by FACS analysis at 2 hours, 4 hours and 6.5 hours post-invasion into the cell line (Figure 6). Fluorescence induced by the various regions of the ssaG promoter were compared to two controls; uninfected J774A.1 cells and J774A.1 infected with S. typhimurium BA275 which contains the promoterless pJKDIO reporter vector. The levels of fluorescence (expressed as the % of cells staining) indicate that all the ssaG promoter fragments tested were capable of inducing GFP expression inside the macrophages in comparison to the controls.
- samples were then stored at -20°C prior to LacZ analysis.
- samples were thawed at room temperature and 144 ⁇ l of each sample placed into a well of a 96-well tissue culture plate (Costar 3590). 16 ⁇ l of 10 x Z buffer (16.1g Na 2 HPO 4 .7H 2 O, 5.5g NaH 2 PO 4 .H 2 0, 0.75g KCI, 0.246g MgSO 4 .7H 2 0, 27 ml ⁇ -Mercapto-ethanol made up to 100 ml with distilled water, pH 7.0) was added.
- the results show that expression of the two reporters is induced inside macrophages by both the 467 bp and 97 bp regions of the ssaG promoter in comparison to the promoterless control vector.
- the expression of the two reporter proteins correlates during the course of the experiment, with maximal expression of LacZ and GFP occurring at 5 hours post-invasion.
- the LacZ experiment shows that there remains a high level of expression of LacZ from both constructs at 24 hours post infection.
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- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02702574A EP1365802A2 (en) | 2001-03-09 | 2002-03-11 | Salmonella promoter for heterologous gene expression |
AU2002236096A AU2002236096A1 (en) | 2001-03-09 | 2002-03-11 | Salmonella promoter for heterologous gene expression |
US10/471,385 US20040131637A1 (en) | 2001-03-09 | 2002-03-11 | Salmonella promoter for heterologous gene expression |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0105924.5A GB0105924D0 (en) | 2001-03-09 | 2001-03-09 | Promoter |
GB0105924.5 | 2001-03-09 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002072845A2 true WO2002072845A2 (en) | 2002-09-19 |
WO2002072845A3 WO2002072845A3 (en) | 2002-11-21 |
Family
ID=9910384
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2002/001098 WO2002072845A2 (en) | 2001-03-09 | 2002-03-11 | Salmonella promoter for heterologous gene expression |
Country Status (5)
Country | Link |
---|---|
US (1) | US20040131637A1 (en) |
EP (1) | EP1365802A2 (en) |
AU (1) | AU2002236096A1 (en) |
GB (1) | GB0105924D0 (en) |
WO (1) | WO2002072845A2 (en) |
Cited By (1)
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WO2010017383A1 (en) | 2008-08-06 | 2010-02-11 | Emergent Product Development Uk Limited | Vaccines against clostridium difficile and methods of use |
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Citations (2)
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WO2000014240A2 (en) * | 1998-09-04 | 2000-03-16 | Creatogen Aktiengesellschaft | Attenuated salmonella sp12 mutants as antigen carriers |
WO2000068261A2 (en) * | 1999-05-10 | 2000-11-16 | Microscience Limited | Attenuated microorganisms for the treatment of infection |
-
2001
- 2001-03-09 GB GBGB0105924.5A patent/GB0105924D0/en not_active Ceased
-
2002
- 2002-03-11 WO PCT/GB2002/001098 patent/WO2002072845A2/en not_active Application Discontinuation
- 2002-03-11 EP EP02702574A patent/EP1365802A2/en not_active Withdrawn
- 2002-03-11 US US10/471,385 patent/US20040131637A1/en not_active Abandoned
- 2002-03-11 AU AU2002236096A patent/AU2002236096A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000014240A2 (en) * | 1998-09-04 | 2000-03-16 | Creatogen Aktiengesellschaft | Attenuated salmonella sp12 mutants as antigen carriers |
WO2000068261A2 (en) * | 1999-05-10 | 2000-11-16 | Microscience Limited | Attenuated microorganisms for the treatment of infection |
Non-Patent Citations (5)
Title |
---|
CIRILLO DANIELA MARIA ET AL: "Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival." MOLECULAR MICROBIOLOGY, vol. 30, no. 1, October 1998 (1998-10), pages 175-188, XP002212516 ISSN: 0950-382X * |
DUNSTAN SARAH J ET AL: "Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium." INFECTION AND IMMUNITY, vol. 67, no. 10, October 1999 (1999-10), pages 5133-5141, XP002212811 ISSN: 0019-9567 * |
HENSEL MICHAEL ET AL: "Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages." MOLECULAR MICROBIOLOGY, vol. 30, no. 1, October 1998 (1998-10), pages 163-174, XP002212433 ISSN: 0950-382X cited in the application * |
SHEA JACQUELINE E ET AL: "Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 93, no. 6, 1996, pages 2593-2597, XP002213357 1996 ISSN: 0027-8424 * |
VALDIVIA RAPHAEL H ET AL: "Fluorescence-based isolation of bacterial genes expressed within host cells." SCIENCE (WASHINGTON D C), vol. 277, no. 5334, 1997, pages 2007-2011, XP002212432 ISSN: 0036-8075 cited in the application * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010017383A1 (en) | 2008-08-06 | 2010-02-11 | Emergent Product Development Uk Limited | Vaccines against clostridium difficile and methods of use |
EP2318515A1 (en) * | 2008-08-06 | 2011-05-11 | Emergent Product Development UK Limited | Vaccines against clostridium difficile and methods of use |
EP2318515A4 (en) * | 2008-08-06 | 2012-11-14 | Emergent Product Dev Uk Ltd | Vaccines against clostridium difficile and methods of use |
Also Published As
Publication number | Publication date |
---|---|
EP1365802A2 (en) | 2003-12-03 |
US20040131637A1 (en) | 2004-07-08 |
WO2002072845A3 (en) | 2002-11-21 |
GB0105924D0 (en) | 2001-04-25 |
AU2002236096A1 (en) | 2002-09-24 |
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